Proteins play an important role in today's medical portfolio. For human application every pharmaceutical substance has to meet distinct criteria. To ensure the safety of biopharmaceutical agents to humans nucleic acids, viruses, and host cell proteins, which would cause severe harm, have to be removed especially. To meet the regulatory specification one or more purification steps have to follow the manufacturing process.
Recombinant polypeptides can be produced e.g. by prokaryotic cells such as E. coli. The recombinantly produced polypeptide accounts for the majority of the prokaryotic cell's polypeptide content and is often deposited as insoluble aggregate, i.e. as a so called inclusion body, within the prokaryotic cell. For the isolation of the recombinant polypeptide the cells have to be disintegrated and the recombinant polypeptide contained in the inclusion bodies has to be solubilized after the separation of the inclusion bodies from the cell debris. For the solubilization chaotropic reagents, such as urea or guanidinium chloride, are used. To cleave disulfide bonds reducing agents, especially under alkaline conditions, such as dithioerythritol, dithiothreitol, or β-mercaptoethanol are added. After the solubilization of the aggregated polypeptide the globular structure of the recombinant polypeptide, which is essential for the biological activity, has to be reestablished. During this so called renaturation process the concentration of the denaturing agents is (slowly) reduced, e.g. by dialysis against a suited buffer, which allows the denatured polypeptide to refold into its biologically active structure. After renaturation the recombinant polypeptide is purified to a purity acceptable for the intended use. For example, for the use as a therapeutic protein a purity of more than 90% has to be established.
Recombinantly produced polypeptides are normally accompanied by nucleic acids, endotoxins, and/or polypeptides from the producing cell. Beside the host cell derived by-products also polypeptide-derived by-products are present in a crude polypeptide preparation. Among others shortened variants of the polypeptide of interest can be present.
In WO 95/25786 the production of human apolipoprotein A1 in a bacterial expression system is reported.